枣自交与异交花粉管生长行为及早期胚胎发育研究

Observation of pollen tubes behavior and early embryogenesis following self and cross-pollination in Chinese jujube

【目的】比较不同枣品种自交与异交授粉后花粉管在花柱组织中生长行为及早期胚胎发育的差异,明确枣自交不亲和性的特征及类型,完善枣生殖生物学,进而从自交不亲和性方面探明枣低含仁率问题,为枣果实生殖学和枣树杂交育种提供理论基础。【方法】设计自交授粉组合‘冬枣’和‘灰枣’以及异交授粉组合‘蜂蜜罐’ב襄汾圆枣’‘、蜂蜜罐’ב延川老牙酸枣’‘、灰枣’ב晋枣’‘、襄汾圆枣’ב永济蛤蟆枣’。根据枣雌蕊特点,经改良软化法处理后,综合运用花粉管荧光显微及石蜡切片观察授粉后自交与异交授粉组合花粉管在雌蕊中的生长及早期胚胎发育情况,从解剖学角度研究枣自交不亲和性。【结果】枣(品种)自交与异交授粉后3 h和6 h均观察到花粉粒在柱头上的萌发,花粉管顺利穿透乳突细胞侵入柱头,向花柱通道内生长。自交授粉组合‘冬枣’和‘灰枣’的花粉管授粉后24 h,生长至花柱三分之一处;授粉后48 h,花粉管生长至花柱二分之一处;授粉后72 h、96 h花粉管处于停滞状态,保持在花柱组织二分之一处。异交授粉组合‘蜂蜜罐’ב襄汾圆枣’‘、蜂蜜罐’ב延川老牙酸枣’‘、灰枣’ב晋枣’‘、襄汾圆枣’ב永济蛤蟆枣’授粉后24 h,花粉管生长至花柱二分之一处;授粉后48 h,花粉管生长至花柱的基部;授粉后72 h,花粉管穿透花柱基部,到达胚珠。授粉后13 d‘,蜂蜜罐’ב襄汾圆枣’的胚胎发育正常,胚胎发育处于合子横向分裂后向小球形胚发育阶段,胚乳的发育则处胚乳游离核时期。但‘灰枣’自交授粉组合表现出由于传粉受精不良导致的胚败育现象。【结论】解剖研究发现枣自交不亲和反应发生在花柱组织内,其类型属配子体自交不亲和中的合子前不亲和类型;枣自交不亲和性与胚败育共同导致枣果实的低含仁率和低坐果率问题,严重制约枣树的杂交育种工作。

【Objective】Ziziphus is an important genus within the family Rhamnaceae.This genus includes several important fruit tree species that are widely planted in China and India,such as the Chinese jujube(Ziziphus jujuba Mill.),the wild jujube(Z.acido jujuba),and the Indian jujube(Z.maur-itiana).Chinese jujube,native to China and domesticated from wild jujube,is one of the most important and widely dried fruit crops in China.But wide spread of blossom and fruit drop,difficulty in hand emasculation,low fruit set,low seed kernel and self-incompatibility seriously restricted the germplasm innovation and hybrid breeding of jujube,and there is an urgent need to promote crossbreeding in production.Therefore,to investigate difference in the growth behavior of pollen tube in the pistil and early embryogenesis of Chinese jujube after self-and cross-pollination,to identify the types and characteristics of self-incompatibility of the plant and to complement the current knowledge of Chinese jujube in reproductive biology.Further to analyze the problems of low kernel content in terms of self-incompatibility and also to provide a theoretical basis for reproduction and cross-breeding.【Methods】The pollination materials are five to eight years general Zizyphus jujube Mill.‘Dongzao’,‘Fengmiguan’,‘Xiangfenyuanzao’,‘Huizao’,‘Yongjihamazao’,‘Yanchuanlaoyasuanzao’,‘Jinzao’.Designing self-pollinators of‘Dongzao’,‘Huizao’and cross-pollinators include‘Huizao’בJinzao’,‘Fengmiguan’בXiagfenyuanzao’,‘Xiangfenyuanzao’×’Yongjihamazao’,‘Fengmiguan’בYanchuanlaoyasuanzao’.According to the experimental principles of observation of the pollen tube under a fluorescence microscope as well as the characteristics of the pistil,the growth process of pollen tube in the pistil and early embryogenesis were observed using fluorescence microscopy combined with the modified softening method,and the routine paraffin method,to characterize the anatomic properties of the self-incompatibility of Chinese jujube.Operation is expressed in the following way:the pollinated flowers tagged and recovered immediately with paper bags,pistils collected at 3 h,6 h,24 h,48 h,72 h,96 h after pollination and immersed in FFA fixative solution,fixed 24 h after vacuum pumping for 20 minutes,then flushed with distilled water and transferred into 70%ethanol solution,stored at 4℃.Hand-pollinated pistils were hydrated with an ethanol series(70%,50%and 30%ethanol)for 30 min,washed with distilled water,and boiled with 2 mol·L-1 of sodium hydroxide solution for 30 min to allow softening,washed with distilled water again,immersed in buffer solution(Glacial acetic acid and sodium hydroxide configuration,pH 5.5 to 6.5)for 30 min and stained overnight with 0.1%(m/V)aniline blue solution containing 108 mmol·L-1 K3 PO4 at pH 11 and 2%glycerol after washing with distilled water.Pistil was sucked with plastic drop pipette,and dropped on glass slides,covered with cover glass lightly without pressing.The section under the OlympusBX-51 fluorescence microscope was used to observe the pollen germination and pollen tube growth in the style,and take pictures.According to the results of pollen tube growth,flowers were selected for paraffin section,processes as follows:fixation,dehydration,clearing,wax-filling,embedding and trimming,sectioning,adhering to slides,deparaffinization and staining,mounting,slice thickness 6 to 8μm which was stained with Safranine and Fast Green.Olympus microscope imaging system was used to observe the structure and formation of the embryo.【Results】In self and cross-pollination of different jujube cultivars,Pollen grains begun to germinated on stigma,3 h AFP;Pollen tube penetrated into stigma and grew into stylar canal,6 h AFP.pollen tubes penetrated at 1/3 of stylar canal,24 h after pollination;pollen tubes penetrated at middle of stylar canal,48 h after pollination;pollen tube that stopped growing while penetrated at middle of stylar canal,96 and 129 h after pollination as was observed in the pollination combination(‘Dongzao’בDongzao’,‘Huizao’בHuizao’).In pollination combination(‘Huizao’בJinzao’,‘fengmiguan’בXiangfenyuanzao’,‘Xiangfenyuanzao’בYongjihamazao’),pollen tubes penetrated at 1/2 of stylar canal,24 h after pollination;pollen tubes grew to the lowest part of stylar canal,48 h after pollination;pollen tubes penetrated the stylar base and reached the ovule,72 h after pollination.13 days after pollination,microscopic observation showed that in the ovary of pollination(‘fengmiguan’בXiangfenyuanzao’),one ovule degenerated and the other kept on developing.Globular stage gradually formed after the transverse fissure of zygote,free nucleus of endosperm gradually increased.Embryo abortion caused by poor pollination and fertilization was observed in pollination(‘Huizao’בHuizao’).【Conclusion】Based on the anatomical characteristics,we ascertained that Chinese jujube was a self-incompatible plant and belonged to the prezygotic incompatibility type,which occurred in the stylar base.The current study revealed that the Chinese jujube with very low kernel content and fruit set are caused firstly by self-incompatibility and followed by abortion due to hypoplasia of embryo,and this finding also provides insight into crossbreeding and parental choice for the jujube.